Borrelia burgdorferi, the causative agent of Lyme disease, survives and proliferates in both an arthropod vector and various mammalian hosts. During its transmission/infective cycle, B. burgdorferi encounters environmental challenges specific to those hosts. One such challenge comes from reactive oxygen species (ROS) e.g. superoxide radicals (O2.-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH.) and reactive nitrogen species (RNS) e.g. nitric oxide (NO), N2O3 and peroxynitrite. There are two stages in the infective cycle when B. burgdorferi is exposed to ROS/RNS. The first is during the initial stages of infection of the mammalian host when cells of the immune system attempt to limit and eliminate B. burgdorferi using several mechanisms including the production of ROS and RNS. Surprisingly, the second ROS/RNS challenge occurs as the bacteria migrate through the salivary glands during transmission. Our lab, in collaboration with Dr. Tom Schwan, has demonstrated that the salivary glands of Ixodes scapularis contain significant levels of ROS. Therefore, our current working model is that as B. burgdorferi migrates from the anaerobic midgut (containing no ROS) to the salivary glands, ROS act as a signal to induce the expression of ROS defense enzymes and key virulence factors that promote the survival and successful colonization of a new host. Cellular defenses against the damaging effects of ROS involve both enzymatic and nonenzymatic components. B. burgdorferi has a limited number of enzymes that could potentially be involved in this defense response. Those identified include a Mn-dependent superoxide dismutase (SOD), a Dps/Dpr homologue (NapA), thioredoxin (Trx), thioredoxin reductase (TrxR) and a Coenzyme-A disulfide reductase (CoADR). To date the Mn-SOD, CoADR, and NapA (unpublished data) have been characterized experimentally. Because these enzymes would promote the in vivo survival of B. burgdorferi cells when challenged by O2.- and H2O2 from host cells, we are particularly interested in the process and how it is regulated. The Borrelia oxidative stress regulator, BosR, acts as a transcriptional activator of oxidative stress genes. Although there is no apparent amino acid homology (>15%), BosR appears to be functionally similar to OxyR from E. coli. In addition, we have shown that BosR regulates sodA (Mn-SOD), the gene encoding NapA (an AhpR homolog), and cdr (CoADR). The effects of ROS/RNS on cells have been extensively investigated. These highly reactive compounds have been shown to damage cellular macromolecules including DNA, proteins, and membrane lipids. In eukaryotes, membrane lipids are a major target of reactive oxygen species. Free radicals attack polyunsaturated fatty acids in membranes and initiate lipid peroxidation. A primary effect is a decrease in membrane fluidity which affects the physical properties of the membrane altering the function of membrane-associated proteins. Once lipid peroxides form, they react with adjacent polyunsaturated lipids causing an amplification of the damage. Lipid peroxides undergo further oxidation to a variety of products, including aldehydes, which subsequently react with and damage membrane proteins. However, in bacteria, it is assumed that lipids are not subject to the oxidative damage observed in eukaryotic cells. Only certain polyunsaturated lipids, such as linoleic and linolenic acid, are susceptible to oxidation and it is clear that most bacteria do not synthesize or incorporate these types of lipids in their cell membranes. Two notable exceptions are the photosynthetic bacteria and Borrelia species who synthesize or incorporate significant levels of linolenic acid in their membranes. Instead, it has been shown that the most damaging effects of ROS in bacteria result from the interactions of radicals (H2O2) with "free" Fe2+ generating very reactive OH- (Fenton reaction). Because of this reactivity, its effect on any given biomolecule will depend largely upon proximity to the target. Because Fe2+ localizes along the phosphodiester backbone of nucleic acid, DNA is a major target of OH-.This reactive species can pull electrons from either the base and sugar moieties producing a varety of lesions including single and double strand breaks in the backbone and chemical crosslinks to other molecules. These strand breaks, and other lesions that block DNA replication, contribute to OH.- toxicity and cell death. Other base damage, that does not hinder replication, contributes to a significant increase in mutation rates. The intracellular biochemistry of B. burgdorferi suggest that the primary intracellular target of ROS may not be DNA as described in other bacteria such as E. coli. In E.coli, the extent of DNA damage due to H2O2 and Fenton chemistry is directly proportional to Fe metabolism and the free Fe concentration within the cell(5-100 nM). Since the intracellular Fe concentrations of B. burgdorferi are estimated to be <10 atoms per cell, it seems unlikely that DNA is a primary target for ROS in B. burgdorferi. In support of this, growth of B. burgdorferi in the presence of 5mM H2O2 had little to no effect on the DNA mutation rate (spontaneous coumermycin A1 resistance). Also, when cells were treated with various oxidants (t-butyl peroxide or H2O2) no increase in DNA damage was detected in comparison to untreated cells as determined by calculating the number of DNA base lesions using an aldehyde reactive probe. As previously mentioned, B. burgdorferi incorporates polyunsaturated fatty acids from the environment into the cells membrane lipids and lipoproteins suggesting that Borrelia membranes could be a target for lipid peroxidation. Analyses of t-butyl peroxide treated B. burgdorferi cells by electron microscopy showed significant irregularities indicative of membrane damage. Fatty acid analysis of cells treated with t-butyl peroxide and lipoxidase indicated that host-derived linoleic acid had been dramatically reduced (10- and 50-fold, respectively) in these cells, with a subsequent increase in the levels of malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) aldehyde(4- and 10-fold respectively), the toxic by-products of lipid peroxidation. These data, taken together, suggest that B. burgdorferi membrane lipids and lipoproteins are the primary targets for attack by ROS. B. burgdorferi is resistant to killing by reactive oxygen species (ROS), yet, previous studies have demonstrated that it is highly susceptible to killing by reactive nitrogen species (RNS). In this study, diethylamine NONOate (DEA/NO) was used to characterize the lethal effects of RNS on B. burgdorferi. RNS produce a variety of lethal DNA lesions, however, levels of the DNA deamination product, deoxyinosine, as well as the number of apurinic/apyrimidinic sites were identical in DNA isolated from untreated and DEA/NO-treated cells. The lack of DNA damage in DEA/NO-treated B. burgdorferi was due, in part to the activity of the Nucleotide Excision Repair (NER) pathway as shown by hypersensitivity of uvrC- and uvrB-deficient strains of B. burgdorferi to RNS, along with an increased spontaneous mutation rate leading to coumermycin A1 resistance. Polyunsaturated fatty acids in B. burgdorferi cell membranes, which are susceptible to peroxidation by ROS, were not sensitive to RNS-mediated lipid peroxidation (assayed by measuring malondialdehyde). However, proteins from DEA/NO-treated B. burgdorferi cells displayed a high degree of S-nitrosylation, as well as increased levels of cytoplasmic zinc (measured with Zinquin) indicative of damage to free and zinc-bound cysteine thiols (e.g., BosR, NapA and fructose-1,6biphoahphate aldolase). These data suggested that proteins with free or zinc-bound cysteine thiols are the major targets of RNS in B. burgdorferi.